Coolable carrier, apparatus and method for producing frozen sample spheres

Abstract

The present invention relates to a coolable carrier with a plurality of receiving structures for receiving and positioning liquid samples, as well as to its use and a method for producing frozen sample spheres. [Solution] The coolable carrier (1) according to the present invention comprises at least one structured surface (2) having a plurality of receiving structures (A), each of which can be used to receive and position a liquid sample (5), and each receiving structure (A) has a recess (3), in particular a concave recess. The carrier (1) is characterized in that at least some of the receiving structures (A) are exposed from the structured surface (2) by protruding beyond the surface (7) of the structured surface (2) that immediately surrounds the receiving structure (A). The present invention also relates to the use of the carrier (1) and to a method for producing frozen sample spheres (10).

Description

[Technical Field]

[0001] The present invention relates to a coolable carrier as defined in the preamble of the main claim, its use, and an apparatus and method for producing frozen sample spheres. [Background technology]

[0002] In the following, the term "cryobeads" refers to spherical frozen solutions (sample spheres) containing various active ingredients, in particular therapeutic active ingredients, excipients, enzymes, proteins, genetic material (especially DNA and / or RNA fragments), primers or oligonucleotides, salts, organic substances, complexes and / or other substances or molecules, vesicles, chromosomes, organelles and complete cells. These are also referred to herein as "reagent mixtures." After their production, cryobeads can be advantageously stored at ambient temperatures above the respective freezing point by removing the water they contain.

[0003] The advantage of such dried cryobeads is that even initially sensitive materials can be transported without necessarily maintaining a cold chain, and therefore can be used in processes and / or laboratories in countries with inadequate or non-existent cooling infrastructure.

[0004] These cryobeads are preferably manufactured in a spherical shape to ensure the best possible mechanical stability and surface area to volume ratio, which makes them and their dried forms (lyobeads) most stable against mechanical stresses, and their compact shape allows for easy transport and use without material loss due to wear, while also saving space.

[0005] Essentially, three methods for producing cryobeads are known from the prior art. In the first method, ice particles, such as dry ice pellets, are compressed using a punch or a combination of a punch and a die to form spheres or pellets. However, this method is technically difficult to control for use in the pharmaceutical and diagnostic fields.

[0006] In the second manufacturing method, the reagent mixture is frozen in or on liquid nitrogen or other cryogenic liquid (e.g., silicone oil). If the droplets of the reagent mixture are small enough, they initially float on the evaporating nitrogen and solidify into spheres due to the "reverse Leidenfrost effect." As the droplets cool, the temperature difference with the still-boiling nitrogen decreases, causing them to sink. If the droplets are too large or heavy, the evaporating nitrogen cannot hold them on the surface. As they sink, they solidify into droplets. This is particularly true for non-boiling cryogenic liquids such as silicone oil.

[0007] The boiling of nitrogen can cause tiny volumes, so-called "satellite droplets," to separate from the still-liquid droplet, leading to an undesirable and uncontrollable decrease in droplet volume. Furthermore, the boiling nitrogen causes droplets suspended on the nitrogen vapor to move back and forth, colliding with other droplets and fusing them into undesirable larger droplets. This results in beads of different sizes, i.e., beads with a non-uniform size distribution. Furthermore, the droplets may come into direct contact with the nitrogen and impurities or substances that may contaminate the sample before completely freezing.

[0008] In a third method, droplets of the solution to be processed to form cryobeads are dispensed onto a (super)cooled plate and frozen there. To facilitate spheronization (i.e., shaping into a spherical shape) of the droplets, the plate can be made hydrophobic or otherwise repulsive in at least the relevant surface area. This method typically produces hemispheres, spherical segments, or flattened ellipsoidal lenses rather than true spheres. The cryobeads formed on the plate must then be mechanically removed from the plate, which can easily damage the individual cryobeads and cause them to lose their desired shape and volume of the reagent mixture.

[0009] Examples of cryobead production and optional drying include those described in US 4,848,094, US 2014 / 0294872 A1, US 2016 / 0252300 A1, WO 2009 / 092703 A1, WO 2010 / 125087 A1, and WO 2013 / 066769 A1. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent No. 4,848,094 [Patent Document 2] U.S. Patent Publication No. 2014 / 0294872 [Patent Document 3] U.S. Patent Publication No. 2016 / 0252300 [Patent Document 4] International Publication No. WO2009 / 092703 [Patent Document 5] International Publication No. WO2010 / 125087 [Patent Document 6] International Publication No. WO2013 / 066769 Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention aims to provide an improved means for producing frozen sample spheres. It is an object of the present invention to provide means relating to an apparatus and method for achieving the above object. [Means for solving the problem]

[0012] The above object is achieved by a carrier and its use according to the main claim and by a device and a method according to the independent claims. Advantageous developments are set out in the dependent claims.

[0013] The coolable carrier has at least one structured surface on which a plurality of receiving structures are formed, each of which functions to receive and position a predetermined amount, e.g., an aliquot, of a liquid sample (hereinafter abbreviated as "sample"). The term "liquid sample" includes not only aqueous solutions of various viscosities but also gels that can form spheres (spherical bodies) under appropriate conditions due to intermolecular interactions. Each receiving structure has a concave recess. The receiving structures have a predetermined receiving capacity for the sample (reagent mixture) and are designed to prevent unwanted spillage or rolling. Characteristically, at least some of the receiving structures protrude above the surface of the immediately surrounding structured surface and are separated from the structured surface. Furthermore, at least some of the receiving structures may optionally be provided with a weak to moderately repulsive, particularly hydrophobic, coating and / or surface structure in the area in direct contact with the sample to promote sphericity and increase the contact angle between the sample and the carrier surface. The dimensions of the recess determine the positioning of the sample, particularly in a liquid state, and constitute a mechanical spatial resistance that prevents the sample from rolling.

[0014] Particularly suitable materials for the carrier are materials that can effectively cool the receiving structure, ie have as high a thermal conductivity as possible, such as metals and metal alloys, composites with a metal layer, etc.

[0015] In a further embodiment of the invention, the region of the structured surface surrounding the recess is also provided with a hydrophobic coating and / or surface design that maximizes the contact angle between the reagent mixture and the surface. In a further embodiment, each recess formed in the structured surface has a peripheral edge around it that protrudes from the structured surface.

[0016] The recess itself advantageously has the shape of a spherical segment to support the formation of a sphere. The spherical segment is characterized by the corresponding radius of the sphere and the height of the spherical segment. If the height of the spherical segment is equal to the radius of the sphere, the spherical segment corresponds to a hemisphere. In a preferred embodiment, the spherical segment is shallower than a hemisphere, i.e., the ratio of the height of the spherical segment to the radius of the sphere is less than 1, which facilitates the removal of the cryobeads. In a particularly preferred embodiment, this ratio is less than 0.7. Even more preferably, the ratio is less than 0.4.

[0017] The receiving structures separated from the structured surface advantageously protrude from the surface of the structured surface that immediately surrounds them by at least 1 nm, preferably at least 0.1 μm, more preferably at least 1 μm.

[0018] For this purpose, recesses or grooves can be formed around the receiving structures using, for example, subtractive processes such as milling or electrical discharge machining (sinking, drilling, wire electrical discharge machining, etc.). Due to the protrusions thus formed, the receiving structures can have a base with a carrier structure and, for example, a columnar form. The columns can, for example, have a circular, elliptical, or n-sided cross section. Due to this protrusion, the receiving structures also protrude from the bottom of the recesses or grooves that immediately surround them. Advantageously, the groove surrounding the single receiving structure has an internal width of the groove wall surrounding said receiving structure that is at least as large as the diameter of the sample sphere to be formed.

[0019] When the receiving structures present on the structured surface are positioned close enough to one another that their respective grooves fully or partially contact or intersect, leaving only the remnants of the termination surfaces and / or groove walls from the original structured surface.

[0020] When the grooves completely intersect, the groove walls are completely removed, leaving only optional edges of the carrier located outside all receiving structures, with the edges and receiving structures protruding at least 1 nm from the bottom of the groove. In other words, in this configuration, the receiving structures are separated from the original surface by the removal process, leaving no secondary structures between or adjacent to them, such as remnants of the groove walls. The optional edges preferably serve to support and guide a scraper (described below), allowing the produced frozen sample spheres to be automatically removed from the carrier.

[0021] Similarly, in other embodiments, some or all of the receiving structures can be formed on the structured surface by additive methods such as 3D printing (SLS, SLA, FDM, etc.) or sputtering, so that they protrude from at least one surface. In such embodiments, the receiving structures also comprise a carrier structure at their base and are particularly designed as pillars. The pillars can have, for example, a circular, elliptical, or n-sided cross section. The receiving structures protrude from the structured surface by at least 1 nm.

[0022] To minimize the risk of two adjacent sample spheres fusing together, the average distance between the two receiving structures (the distance between their imaginary centerlines or longitudinal axes) is advantageously the same as the diameter of the sample sphere to be formed, particularly preferably at least 1.1 times that diameter.

[0023] In each of the above-described embodiments, the ratio of the diameter of the recess of the receiving structure to the diameter of the receiving structure itself is at least 0.7, preferably at least 0.8, and more preferably at least 0.9, to support the formation of sample spheres. This means that the receiving structure or its edge (see above) has only a very narrowly defined area parallel to the structure surface, extending from its outside to the recess, in the area where it protrudes above the surface immediately surrounding it. This design supports the formation of sample spheres by reducing contact with dense, potentially highly wettable materials. In this way, even highly wettable reagent mixtures with small contact angles can be safely retained and frozen within the recess of the receiving structure. Furthermore, a coating with a hydrophobic effect can be applied, for example, to the front surface of the edge or to the wall of the recess.

[0024] The receiving structures can have diameters selected from the range of, for example, 0.1 mm to 20 mm, which allows for the application and processing of liquid samples, for example, from less than 1 μl to 4000 μl. Advantageously, but not exclusively, each carrier comprises only one type and size of receiving structure, in order to allow for efficient production of frozen sample spheres.

[0025] It is also possible to provide carriers with different types and / or different dimensions of receiving structures, which can be used, for example, to find, test and, if necessary, optimize the receiving structure suitable for a particular reagent mixture (sample).

[0026] The present invention aims to enable efficient production of large quantities of frozen sample spheres. For this purpose, the receiving structures are preferably arranged on the structured surface of the carrier in a regular pattern, in particular in rows and / or columns. This regular arrangement allows for an advantageous use of the available area of ​​the structured surface, and also makes the positions of the recesses or frozen sample spheres predictable, which allows for handling in a preferably automated manner (see below).

[0027] To efficiently load the carrier with the sample and easily separate ("harvest") the frozen sample spheres, possibly automatically, the carrier of the present invention may be configured, for example, as a plate. To interconnect multiple plate-shaped carriers, the carrier base may be designed as a rod with an n-sided cross section, with at least one of the n sides being a structured surface. At least one plate-shaped carrier may be exchangeably attached to the base. Such a base has multiple structured surfaces that can be controlled and rotated around its longitudinal axis, making it advantageous to supply a specific surface to a predetermined working position. For example, one surface is filled with a liquid sample at that position, while the other surface is simultaneously harvested at that position.

[0028] According to the concept of rotating the surface, the coolable carrier of the present invention can be configured to have a curved structured surface. The curved structured surface can be a part of a cylinder. In order to support the unidirectional movement of the curved carrier from an automation perspective, the curved surface can be the outer surface of a cylinder.

[0029] In addition to the carrier of the present invention, the object of the present invention is also achieved by an apparatus for producing frozen sample spheres (cryobeads). This apparatus includes at least one carrier of the present invention and a cooling device for cooling the carrier. The cooling device can be, for example, an electric Peltier element disposed on the carrier. Alternatively, a refrigerator can transport a refrigerant liquid to the carrier and circulate the refrigerant through cooling channels in the carrier. Cooling with liquid nitrogen or dry ice is also possible. In this case, cooling fins can be provided, particularly on the surface opposite the structured surface (bottom surface), to increase the surface area. A scraper is provided to separate the frozen sample spheres produced during use of the apparatus from the receiving structure. The structured surface and the scraper are arranged to be movable relative to each other, and the scraper can be moved along the fixed surface, the structured surface can be moved relative to the fixed scraper, or both can be moved relative to each other.

[0030] To remove the frozen sample spheres from the recesses without damage, the scraper can be designed with an inclined contact surface, advantageously oriented at an obtuse angle to the direction of relative movement between the structured surface and the scraper, so that the scraper acts like a wedge to lift the frozen sample spheres out of their respective recesses (as will be explained in more detail below).

[0031] In yet another embodiment of the present device, a device for automatically dispensing liquid samples into wells is provided. This device can be configured, for example, as a pipetting head, dispenser, or multi-channel pipetting system (e.g., a 12-channel pipetting system). For example, using a 16 x 24 format pipetting head, 384 wells can be filled simultaneously with liquid samples. Other grids and formats, such as 1 x 8, 8 x 12, and 32 x 48, are also possible. By parallelizing the process in this way, the so-called cycle time required for dispensing each liquid sample can be reduced to a few milliseconds compared to conventional methods, such as dropping liquid samples into liquid nitrogen. The drive of the device, carrier and / or scraper may be controlled by a controller to move or operate in a coordinated manner.

[0032] Preferably, the device can be operated under a controlled gas atmosphere. To this end, the device can comprise a supply for supplying gas and its outlet, and optionally a housing. The supply can be fixedly oriented relative to the carrier or can be arranged movably. A movable supply advantageously allows the outlet to be adjusted for a specific pipetting position. The housing can enclose at least the carrier and can also include a scraper, a pipetting head, and / or a cooling system. The gas is in particular a protective gas or gas mixture (including atmospheric air), and it is desirable that it has as low a water content as possible. The carrier according to the invention or the device according to the invention can be used in a method for producing frozen sample spheres.

[0033] Such a method includes providing a carrier or device according to the present invention. A liquid sample is then delivered to at least one receiving structure. The liquid sample can be delivered, for example, by pipetting or dispensing. The total volume V can be delivered using a pipetting head of appropriate dimensions, for example, by simultaneously dispensing individual volumes corresponding to the number of receiving structures. Alternatively, droplets with droplet volumes TV in the pL, nL, or μL range can be delivered in rapid succession at a frequency f ranging from a few Hz to several hundred kHz. For dispensing, the combination of the repetition frequency f of the droplet volumes TV and the total volume V is selected so that the total delivery time t of the total volume V is in the range t = 0.01 s to t = 60 s, preferably t = 0.5 s to t = 15 s. Other liquid transfer methods, such as ultrasonic transfer, are also contemplated.

[0034] The liquid sample dispensed into each well is powerfully cooled, producing a frozen sample sphere. Temperature control can be achieved using, for example, liquid nitrogen, dry ice, an electric Peltier element, or a coolant supplied by a refrigerator. Temperature control can be achieved by cooling the carrier itself or by cooling via direct contact with an actively cooled base plate. Carriers designed as cylinders or rectangular prisms, in particular, can be temperature-controlled by the action of a coolant (e.g., liquid nitrogen, temperature-controlled silicone oil, etc.) via a refrigerator. The coolant can circulate within the carrier without coming into direct contact with the liquid sample or frozen sample spheres.

[0035] To harvest the frozen sample spheres, they can be removed from their respective receiving structures, such as with a scraper, as needed. In another embodiment, once the sample spheres have frozen, they can be collected by gravity, such as by tilting the carrier. To this end, the recesses can be provided with a coating and / or surface structure that, for example, does not adequately retain the frozen sample spheres, even in an inclined or upside-down position. Once collected, the sample sphere must continue to be cooled to prevent it from increasing in temperature uncontrollably, starting to melt again, or thawing completely.

[0036] The frozen sample spheres thus produced can be stored in a cooled state and used later, but to allow them to be stored, transported and used at temperatures above their respective freezing temperatures, the sample spheres are advantageously dried, in particular freeze-dried. [Effects of the Invention]

[0037] The advantages of the present invention are that the liquid sample and the frozen sample spheres do not come into direct contact with the refrigerant, eliminating contamination. The ability to produce frozen sample spheres with minimal dimensional or volume variations due to the manufacturing process is crucial for the reproducibility of their application, even when the manufactured sample spheres are used dry. As a result, the reject rate is low. The combination of efficient production and a relatively simple manufacturing equipment design makes it possible to produce high-quality frozen sample spheres in a compact, low-cost system.

[0038] The method of the present invention allows for the production of sample spheres ranging in diameter from 0.5 to 20 mm, with volumes ranging from less than 1 μl to 4,000 μl. Experiments have demonstrated the production of thousands of 10 μl mock beads, thousands of 5 μl, 10 μl, and 25 μl RT-qPCR beads, and hundreds of 10 μl LAMP beads, all of which were freeze-dried and functionally tested. Performance parameters after freeze-drying were comparable to those of the standard method. For example, the cycle rate can reach 72,000 per hour, and the production of 125,000 sample spheres takes only 1.75 hours.

[0039] The sample spheres produced according to the invention can be used in the freeze-dried state, particularly as precursors, for example as inactive precursors for detection reactions, ready-to-use reaction mixtures or buffers, for example for RT-qPCR (and other PCR variants), isothermal amplification, immunoassays, enzymes and proteins, binding antibodies, collagen, active pharmaceutical ingredients or therapeutic agents, etc. [Brief explanation of the drawings]

[0040] [Figure 1] 1 is a diagram schematically illustrating a first embodiment of a carrier according to the present invention. [Figure 2] FIG. 2 is a diagram schematically illustrating a second embodiment of a carrier according to the present invention. [Figure 3] FIG. 4 is a diagram schematically illustrating a third embodiment of a carrier according to the present invention. [Figure 4] FIG. 10 is a diagram schematically illustrating a fourth embodiment of a carrier according to the present invention. [Figure 5] FIG. 10 is a diagram schematically illustrating a fifth embodiment of a carrier according to the present invention. [Figure 6] FIG. 10 is a diagram schematically illustrating a sixth embodiment of a carrier according to the present invention. [Figure 7] FIG. 10 is a diagram schematically illustrating a seventh embodiment of a carrier according to the present invention. [Figure 8] FIG. 10 is a diagram schematically illustrating an eighth embodiment of a carrier according to the present invention. [Figure 9] FIG. 13 is a diagram schematically illustrating a ninth embodiment of a carrier according to the present invention. [Figure 10] FIG. 22 is a diagram schematically illustrating a tenth embodiment of the carrier according to the present invention. [Figure 11] 1 is a diagram schematically illustrating an embodiment of an apparatus for producing frozen sample spheres according to the present invention. [Figure 12] FIG. 10 is a diagram schematically illustrating yet another embodiment of the apparatus for producing frozen sample spheres according to the present invention. [Figure 13] 1 shows a schematic flow diagram of one embodiment of the method according to the invention, the illustration is schematic and not to scale, the same reference symbols denote the same technical elements in the different figures. DETAILED DESCRIPTION OF THE INVENTION

[0041] The carrier 1 according to the invention has at least one structured surface 2, in particular a side surface, in which at least one recess 3 is formed, which serves as a receiving structure A. In Fig. 1, for example, a total of nine recesses 3 are shown, arranged in three columns and three rows. The recesses 3 are in particular concavely curved in the material of the carrier 1 and represent, for example, spherical segments.

[0042] In the second embodiment, the recess 3 is surrounded by a rim 4 (see FIG. 2). The recess 3 and the rim 4 together form a receiving structure A. The rim 4 is kept narrow in the radial direction on the end face away from the structured surface 2 in order to reduce the potential wetting surface for the liquid sample 5 supplied to the recess 3 (see FIG. 3). Although FIG. 2 shows only one receiving structure A as an example, the carrier 1 may have multiple such receiving structures A.

[0043] To reduce the potential wetting surface and promote the beading of the liquid sample 5, each recess can be surrounded by a groove 6, for example, formed by localized material removal from the structured surface 2. The receiving structure A thus formed is free-standing and protrudes above the surface 7 immediately surrounding it (the bottom surface of the corresponding groove 6). This protrusion allows the receiving structure A to carry a carrier structure T at its base. Figure 3 shows two types of receiving structures A. The receiving structure A on the right side of the figure remains pillar-shaped and has a relatively small diameter and very shallow recess 3. Such a design is suitable, for example, for small amounts of liquid sample 5 or reagent mixtures with a high tendency to bead.

[0044] A liquid sample 5 is applied to one of the receiving structures A shown on the left side of the figure. Its amount and composition cause the formation of spheres based on intermolecular interactions. This process is further supported by the spherical segment shape of the recesses.

[0045] 4, the individual receiving structures A are positioned very close to one another and the respective grooves 6 are large enough that they partially intersect. By way of example, the diameter D1 of the recess 3 has a ratio to the diameter D2 of the receiving structure A of slightly more than 0.7. The circular end face of the receiving structure A facing away from the remaining surface 7 can optionally be provided with a coating and / or surface structure that repels the liquid sample 5.

[0046] The intersecting grooves 6 are even more pronounced in the fifth embodiment shown in the enlarged detail view (Fig. 5). Only a few pillars with pillow-shaped cross-section remain from the original structure surface 2. The carrier 1 is designed like a plate in SBS format and contains 768 receiving structures A for producing 10 μl frozen sample spheres 5 (see Fig. 3) or cryobeads 10 (see Fig. 11). The receiving structures A are arranged in rows and columns, with adjacent ones in each column offset from each other by a distance corresponding to the distance between the receiving structures A and part of the diameter of the surrounding grooves 6, making efficient use of the available space.

[0047] 6 shows a sixth embodiment of the carrier 1 according to the invention, in which the receiving structures A are similarly arranged in rows and columns and offset from one another, but the grooves 6 partially intersect one another. The number of receiving structures A in this example is 192. In a seventh embodiment (FIG. 7) based on the same basic configuration, 384 receiving structures A are provided.

[0048] In contrast to this, in the eighth embodiment (FIG. 8), the grooves 6 of the 384 receiving structures A do not intersect with each other, but are arranged in rows and columns, but are not offset from each other.

[0049] Similarly, as shown in the ninth embodiment (FIG. 9), the receiving structures A or recesses 3 provided on the structure surface 2 of the plate-shaped carrier 1 are not offset from one another. The carrier 1 is provided with a large number of receiving structures A of different designs, and the receiving structures A in two adjacent rows have the same shape. For clarity, the receiving structures A are classified into three regions S1 to S3 in the row direction. The receiving structures A in the first section S1 shown on the far right of the figure are formed as concave recesses 3 in the structure surface 2.

[0050] To the left are the receiving structures A of the second section S2, each surrounded by a groove 6, which does not intersect with one another. The ratio of the diameter of the receiving structures A to the grooves 6, which have a constant diameter, varies every two rows, and each receiving structure A has a very shallow recess 3.

[0051] In the third section S3, the receiving structures A are also surrounded by grooves 6. The ratio of the diameter of the receiving structures A to the constant diameter grooves 6 increases towards the left, i.e. every two rows the diameter of the receiving structures A decreases. The recesses 3, provided on the front side facing the viewer, are concave and in particular have the form of spherical segments.

[0052] A carrier 1 designed according to the ninth embodiment can be advantageously used to determine the appropriate receiving structure A for a particular reagent mixture and / or the desired volume of frozen sample spheres 10 to be produced. This is preferably done while taking into account the interaction with the respective production conditions, such as the particular temperatures of the carrier 1 and liquid sample 5, the time required for the sample spheres to form and transition to a fully frozen state, etc.

[0053] The plate-shaped carrier 1 described above can be used in manual or automated manufacturing methods for producing frozen sample spheres 10. To facilitate continuous supply of liquid sample 5 to the carrier 1 and harvesting of the frozen sample spheres 10, in a tenth embodiment, the carrier 1 is formed as a roller-shaped or n-sided base 11. FIG. 10 shows a carrier 1 according to the tenth embodiment, which includes a roller-shaped base 11, the outer periphery of which is formed by a structured surface 2. The receiving structures A are arranged in rows, and in the illustrated embodiment, adjacent rows are offset from each other to make better use of the available area.

[0054] The roller-shaped carrier 1 is arranged in a device 8 (see also FIG. 11). The supply of the liquid sample 5 to the receiving structure A can be carried out by a device 13 designed for this purpose, which can be configured for example as a dispenser or pipetting head 17.

[0055] One embodiment of an apparatus 8 for producing frozen sample spheres 10 is shown in simplified form in FIG. 11. The carrier 1 is brought to and maintained at the desired temperature by a cooling device 9. A liquid sample 5 is delivered to the corresponding recess 3 (see above) of the receiving structure A by a device 13 configured as a pipetting head 17 for delivering the liquid sample 5, where it freezes. The frozen sample spheres 10 produced in this way are removed from the recess 3 by a scraper 14. This can be done purely mechanically by the pressing force transmitted by the scraper 14 to the frozen sample sphere 10. If the recess 3 is formed as a spherical segment, in particular as a (nearly) hemispherical segment, an inclined scraper 14 is advantageous. FIG. 11 shows the scraper 14, whose contact surface 15 is inclined with respect to its movement relative to the carrier 1. An existing controller 12 is data-transmittingly connected to the pipetting head 17, the drive 16 for the scraper 14, and the cooling device 9 and operates them with control commands.

[0056] In the example, the scraper 14 is guided from left to right against the frozen sample sphere 10. The contact surface 15 of the scraper 14 is inclined to the left and acts like a wedge to lift the frozen sample sphere 10 without damage from the corresponding recess 3. The relative movement between the carrier 1 and the scraper 14 is generated by a drive 16.

[0057] Furthermore, the device 8 of Figure 11 comprises a supply 19 for supplying protective gas to the carrier 1. For optimal maintenance of a controlled gas atmosphere, the device 8 also comprises a housing 18.

[0058] In the embodiment of FIG. 12, the device 8 is combined with a coolable roller-shaped carrier 1 (see FIG. 10). The roller-shaped carrier 1 rotates one position each time a liquid sample drop 5 is dispensed from a receiving structure A arranged in a row on a curved structured surface 2. After a rotation of ≥ 90° to < 360°, preferably ≥ 180° to < 360°, and ideally 270°, the sample sphere 10, which has already been dispensed and frozen, reaches a preferably fixed scraper 14. As the carrier 1 rotates further, the sample sphere 10 is pressed against the scraper 14 and removed, i.e., harvested, from the recess 3. In this embodiment, the contact surface 15 of the scraper 14 is also inclined.

[0059] The harvested frozen sample spheres 10 can be placed in a collection container for further processing or storage, and are then advantageously freeze-dried (lyophilized) to allow the frozen sample spheres 10 to be stored, transported, and used without additional cooling.

[0060] An embodiment of the method according to the invention is shown in a simplified representation in Figure 13. After providing at least one carrier 1 according to the invention, it is cooled to a processing temperature at which the reagent mixture used freezes. The processing temperature is usually in the range of 10 K to 350 K, advantageously 75 K to 277.15 K. It is considered advantageous that the temperature of the carrier 1 is within ±50 K of the melting point Tm or eutectic point Teut or collapse temperature TC of the reagent mixture.

[0061] A predetermined amount of liquid sample 5 is supplied to the recess 3 of each receiving structure A of the carrier 1, which is adjusted to the desired processing temperature. There, each predetermined amount of liquid sample 5 forms a sphere due to the intermolecular interactions of its components and is then frozen. The frozen sample spheres 10 thus produced are harvested and further dried as required. [Explanation of symbols]

[0062] 1. Career 2 Structural aspect 3 recess 3a End face 3b Career Structure 4 Edge 5 Liquid samples 6 grooves 7 Groove bottom, remaining surface 8 equipment 9 Cooling device 10 Frozen sample balls 11 Base 12 Controllers 13 Equipment 14 Scraper 15 Contact surface 16 Drive unit 17 Pipetting Head 18 Housing 19 (protective gas) supply section A. Receptor structure D1 Diameter of recess 3 D2 diameter of receiving structure 3 T Carrier Structure

Claims

1. A coolable carrier (1) having at least one structural surface (2), The structured surface (2) comprises a plurality of receiving structures (A), Each receiving structure (A) can be used to receive and position a liquid sample (5); Each receiving structure (A) has a recess (3), in particular a concave recess, At least some of the receiving structures (A) are exposed from the structure surface (2) by protruding from the surface (7) of the structure surface (2) that directly surrounds each receiving structure (A), Coolable carrier.

2. At least some of the receiving structures (A) include a carrier structure (T) disposed at the base thereof; A coolable carrier (1) according to claim 1.

3. A groove (6) is formed in the structure surface (2) around at least a portion of the receiving structure (A). A coolable carrier (1) according to claim 1 or 2.

4. a recess (3) is formed in the end face (3a) of each receiving structure (A), Coolable carrier (1) according to claim 2 or 3.

5. characterised in that the recess (3) has the shape of a spherical segment, A coolable carrier (1) according to any preceding claim.

6. The receiving structure (A) has a diameter selected from the range of 0.1 mm to 20 mm. A coolable carrier (1) according to any preceding claim.

7. characterised in that the ratio between the diameter of the recess (3) and the diameter of the receiving structure (A) is at least 0.7, preferably at least 0.8, more preferably at least 0.9; A coolable carrier (1) according to claim 5.

8. The receiving structures (A) are formed in rows and / or columns on the structure surface (2), A coolable carrier (1) according to any preceding claim.

9. The carrier (1) is configured in a plate shape. A coolable carrier (1) according to any preceding claim.

10. The base (11) of the carrier (1) is configured in a rod shape having an n-sided cross section, At least one of the n sides is a structured surface (2), A coolable carrier (1) according to any preceding claim.

11. The carrier (1) is configured to have a curved structured surface (2), A coolable carrier (1) according to any one of claims 1 to 9.

12. A carrier (1) according to any of the preceding claims, a cooling device (9) for cooling the carrier (1); and a scraper (14) with an inclined contact surface for scraping off the frozen sample balls (10) present on the receiving structure (A). Equipped with The carrier (1) and the scraper (14) are movable relative to each other. A manufacturing device (8) for frozen sample spheres (10).

13. For operation under a controlled gas atmosphere, a gas supply port is provided; Optionally, the device comprises a housing surrounding at least the carrier (1), 13. Apparatus (8) according to claim 12.

14. Use of a carrier (1) according to any one of claims 1 to 10 in a method for producing frozen sample spheres (10).

15. The following steps: providing a carrier (1) according to any one of claims 1 to 11; supplying a liquid sample (5) to at least one cooled receiving structure (A); freezing the liquid sample (5) to produce a frozen sample sphere (10); Optionally, scraping the frozen sample ball (10) from the receiving structure (A); A method for producing a frozen sample sphere (10) comprising:

16. characterised in that the sample balls (10) are dried, in particular freeze-dried, 16. The method of claim 15.

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